Parathyroid hormone/parathyroid hormone-related protein receptor ...

Parathyroid hormone/parathyroid hormone-related protein receptor expression in nude mice with a transplantable canine apocrine adenocarcinoma (CAC-8) and humoral hypercalcaemia of malignancy A

Gr\l=o"\ne,L K

McCauley,

C C

Capen and

T

J Rosol

Department of Veterinary Biosciences, The Ohio State University, Columbus, Ohio, University of Michigan School of Dentistry, Ann Arbor, Michigan, USA (Requests for offprints should be addressed to j Rosol)

USA and

'Department of Periodontics/Prevention/Geriatrics,

Abstract The effect of humoral

(HHM)

related

on

hypercalcaemia of malignancy parathyroid hormone/parathyroid hormone-

protein (PTH/PTHrP) receptor expression

investigated

in nude mice with subcutaneous

was

transplan-

tation of an adenocarcinoma line (CAC-8) which produces PTHrP. Serum calcium and PTHrP concentrations were analysed by colorimetric assay and a two-site IRMA respectively. Mice were hypercalcaemic (3\m=.\3\m=+-\0\m=.\1mmol/l) compared with non-tumour-bearing control mice (2\m=.\1\m=+-\0\m=.\1mmol/l) and had elevated serum PTHrP concentrations (30\m=.\4\m=+-\3\m=.\4pmol/l) compared with non\x=req-\ tumour-bearing control mice (0\m=.\7 \m=+-\0\m=.\1pmol/l. Lumbar vertebrae were analysed by histomorphometry. Tumourbearing mice had a significant (P3 mmol/1) over a 2—3 week period. Terminal serum calcium concentrations are reported. Serum PTHrP concentrations were deter¬ mined by two-site IRMA (Radioisotopic Assay PTHrelated Peptide, Nichols Institute Diagnostics, San Juan Capistrano, CA, USA). Both assays were completed in duplicate with the exception of two hypercalcaemic mice which had serum for only single replicates in the PTHrP immunoassay (200 µ serum per sample).

Mice

PTH/PTHrP receptor mRNA

Nude mice (HSD:athymic nude, 4 weeks old) were obtained from Harlan Sprague-Dawley (Indianapolis, IN,

The calvarium, humérus and right kidney from nude mice were frozen in liquid N2. Total RNA was isolated from frozen tissues by the method of Chomczynski & Sacchi (1987) and quantified by spectrophotometry. Total RNA

USA) and were housed under barrier conditions for 7 days before injection of tumour tissue. Cryopreserved tumour tissue (6—10 mm") was injected subcutaneously between the scapulae. Mice were killed when they had been hypercalcaemic for 2—3 weeks (>3 mmol/1) and the tumour volumes were between 1*0 and 2-0 cm Nude mice were injected with calcein (15 mg/kg, dissolved in saline with 2% NaHC03) 1 day before euthanasia to label actively mineralizing bone surfaces. .

Bone

histomorphometry

Lumbar vertebrae were trimmed of musculature and lateral vertebral processes and fixed for 24 h in 10% buffered formalin at 4 °C. One vertebral body was decal¬ cified in formalin containing 10% EDTA, infiltrated in

JB4 glycol methacrylate (Polysciences Inc., Warrington, PA, USA) for 6 days and embedded as described by Brinn & Pickett (1979). Midline sections were stained for tartrate-resistant acid phosphatase (Sigma Diagnostics, St Louis, MO, USA) and counterstained with haematoxy¬ lin (Rosol et al. 1986). Acid phosphatase-positive osteo¬ clasts had

intensely

red-stained

cytoplasm

and lined the

(20 pg)

expression

electrophoresed on an agarose—formaldehyde to nylon membranes (Duralon U.V., La Inc., Jolla, CA, USA) and hybridized to a Stratagene [32P]dCTP-labelled cDNA probe (R15B) encoding the rat PTH/PTHrP receptor (McCauley et al. 1994). The blots were also hybridized to a cDNA probe for ribosomal 18S RNA as a loading control. The hybridized probes were quantified using Instantlmager Electronic Auto¬ radiography (Packard, Meriden, CT, USA). Data were expressed as ratio of signal intensity for the PTH/PTHrP gel,

was

transferred

to the ribosomal 18S RNA. In addition, bands visualized by autoradiography for 24—72 h using Kodak X-OMAT AR film (Kodak, Rochester, NY,

receptor

were

USA). PTH/PTHrP receptor immunohistochemistry Kidneys and vertebral bodies were stained for PTH/ PTHrP receptor protein by immunohistochemistry by following a protocol obtained from D A Lazowski, Elborn College, London, Ontario, Canada with minor

modifications. The left kidneys were frozen in liquid N2 and embedded in OCT compound (Miles Inc., Elkhart, IN, USA). Sections were cut at 6 µ , thawed and fixed in 10% neutral-buffered formalin for 30 min. Endogenous peroxidase was blocked with 2% H202 in 100% methanol for 15 min, followed by two washes in 100% methanol. The slides were then incubated in antigen-retrieval sol¬ ution (Antigen Retrieval Citra Solution; BioGenex, San Ramon, CA, USA) in a H2500 Microwave Processor (Energy Beam Science Inc., Agawam, MA, USA) at 80 °C for 10 min. Slides were allowed to cool to room tempera¬ ture (RT) in the antigen-retrieval solution before they were washed twice in 100% methanol (5 min), twice in 100% ethanol (5 min) and once in 75% ethanol. After two washes in PBS for 5 min, slides were incubated in 0-3% Triton X-100 for 10 min and washed twice in PBS for 5 min. Non-specific binding was blocked by incubating in 5% fetal bovine serum (FBS) (Gibco-BRL, Gaithersburg, MD, USA) in 10 mM Tris-HCl, pH 7-6, containing 500 mM NaCl and 0-001% Tween (FBS/TBS wash buffer). The primary antibody, polyclonal rabbit anti-PTH receptor (BAbCo, Richmond, CA, USA) was diluted (1:100) into primary antibody diluent (Research Genetics, Huntsville, AL, USA) and applied to the slides overnight at 4 °C. The slides were washed in FBS/HSTBS 5 times for 6 min and the secondary antibody (biotinylated goat anti-rabbit immunoglobulin G (Zymed Laboratories Inc., South San Francisco, CA, USA); 1:5000) was applied for 30 min at RT followed by 5 washes for 6 min in FBS/HSTBS. Slides were incubated with the avidinbiotin complex (ImmunoPure Ultra-Sensitive ABC Staining Kit, Pierce, Rockford, IL, USA) for 30 min, washed in FBS/HSTBS 5 times for 6 min and stained with diaminobenzidine (Research Genetics) until colour

development (3-5 min). The protocol for the

bone sections was modified as follows. The vertebral bodies were decalcified in formalin containing 10% EDTA, embedded in paraffin, sectioned at 6 µ , deparaffimzed in xylene and 100% ethanol, before blockage of endogenous peroxidase. The antigen-retrieval solution (Antigen Retrieval Decal Solution; BioGenex) was applied for 30 min at RT. Controls consisted of slides stained with non-relevant immunoaffinity-purified rabbit immunoglobulin (against glial fibrillary acidic protein; Sigma ImmunoChemicals, St Louis, MO, USA) or omis¬ sion of the primary antibody. The slides were evaluated qualitatively for the presence of positive immunostaining. Statistics

Serum calcium, PTHrP concentrations, bone histomorphometry and image analysis data were analysed by unpaired i-test or Mann-Whitney U test using the statistical pro¬ gram InStat (GraphPad Software version 2-02). Values are presented as mean ± s.e.m. and significance reported at P